ACTIVATED MEMORY T CELLS PRODUCE LIGANDS THAT CAUSE NF-κB-DEPENDENT INFLAMMATORY ACTIVATION OF THE ENDOTHELIUM: IDENTIFICATION OF NOVEL THERAPEUTIC TARGETS

Background: Endothelial cells (EC) are important contributors to inflammation via expression of inflammatory mediators, including cytokines, chemokines and adhesion molecules. Production of these inflammatory mediators can be induced via canonical and NF-κB-inducing kinase (NIK)-dependent noncanonical NF-κB signalling. The ligands activating these pathways are well studied, but less is known about the cells producing ligands that can activate NF-κB signalling in EC. Objectives: To study the effects of factors produced by activated memory T (T m ) cells on NF-κB dependent inflammatory activation of EC. Methods: CD4 CD45RO memory T cells were isolated from healthy PBMC using MACS sorting and cultured in presence of anti-CD3 and anti-CD28 for 72h, after which supernatant was harvested. Endothelial cells were stimulated for 72h with 50% T m supernatant (T m sup) after which protein and RNA was harvested followed by analysis of NF-κB signalling and downstream expression of inflammatory mediators using qPCR and western blot. Culture supernatants were analysed by ELISA for various inflammatory mediators. To repress canonical NF-κB signalling an inhibitor of IKKβ (iIKKβ) was used and to repress NIK-dependent NF-κB signaling an inhibitor of NIK (iNIK) was used in the EC cultures. Results: Stimulation with T m sup led to activation of both canonical NF-κB signalling (increased levels of phosphorylated IκBα) and noncanonical NF-κB signalling (increased p100 to p52 processing). After stimulation with T m sup EC had increased mRNA levels of all tested inflammatory mediators compared to non-treated cells. Gene expression of chemokines, cytokines, and growth factors (CXCL1, CXCL5, IL6, IL8 and GM-CSF) in T m sup stimulated EC was significantly reduced after treatment with iIKKβ and to a lesser, but still significant, extent after treatment with iNIK. Treatment with iIKKβ also led to a reduction in mRNA levels of the adhesion molecules VCAM-1 and ICAM-1, while this effect was less pronounced after iNIK treatment. Of note, treatment with either IKKβ or iNIK led to a significant reduction of CXCL5 in the culture supernatant of T m sup stimulated EC. Conclusion: This study provides new insights into the cellular interactions leading to production of inflammatory mediators by EC. Our findings demonstrate that activated T m cells produce factors that can cause NF-κB-dependent inflammatory activation of EC. Targeting canonical NF-κB signaling via IKKβ or NIK-dependent NF-κB signaling reduces inflammatory activation of the endothelium and may be a potential novel therapeutic target. Disclosure of Interests: None declared DOI: 10.1136/annrheumdis-2019-eular.2979Citation: Ann Rheum Dis, volume 78, supplement 2, year 2019, page A285Session: Cytokines and inflammatory mediators (Scientific Abstracts)